Proteomics can be defined as the comprehensive study of proteins and their functional aspects. Proteins perform the work of the cell. Single proteins can have many forms. The function of a protein depends on the form, interactions, and complexes of the protein. A deeper understanding of the biological functions of proteins is needed so that drugs can be developed.
Protein sample processing is a complex problem within proteomics. Proteins can function individually or as complexes (groups of proteins bound as a complex). Proteins cannot be amplified, as DNA is amplified with polymerase chain reaction (PCR) methods. Proteins must be enriched and purified before they can be analyzed. Protein processing methods and systems must be flexible; more than a million possible proteins are expressed. For analysis it is necessary to separate and concentrate the proteins of interest from many thousands of other proteins, while selectively removing other materials that will interfere with the protein analytical process including cellular material such as other proteins, sugars, carbohydrates, lipids, DNA, RNA and salts. Reproducible recovery is needed and in most cases protein function must be retained during processing. Structural differences between forms must be preserved and final processing of samples must be easily integrated into many different detection schemes, for example mass spectrometry, protein chips, and the like.
Solid phase extraction is one of the primary tools for preparing protein samples prior to analysis. The method purifies proteins according to their identity, class type or structure, or function to prepare them for analysis by mass spectrometry or other analytical methods.
The process of solid phase extraction uses an extraction phase in the form of a column or bed, and the sample may be either loaded onto the column or added to a bulk solution to extraction beads. The extraction phase retains the sample protein, the extraction phase is washed to remove contaminants, and then the sample protein is removed with the extraction or recovery solvent.
Extraction columns are used to prepare the protein samples for analysis. Often very low amounts of proteins are expressed in a sample, and sample preparation procedures are needed to isolate and recover the protein before analysis.
The solid phase extraction of biomolecules such as nucleic acids and proteins is commonly performed by columns packed with a variety of extraction phases.
The need for biomolecule extraction for proteins is increasing rapidly. Large numbers of samples need to be analyzed by a variety of techniques to determine the function of proteins. Typical sample volume is 0.5 to 5 mL or more on a typical column bed volume of 1 to 5 mL, requiring a typical desorption solvent volume of 2 to 10 mL.
There are a number of companies that have developed products whose principle aim is the purification of certain proteins or protein classes by solid phase extraction. The intent of these products is the simplification of proteomic analyses by providing a sample of only those proteins in which the investigator is interested. These products are often packaged for a single use and disposal. Packed-bed columns operate at relatively low pressures, thus making them simple to operate in a highly parallel and automated manner. Due to the very nature of a conventional packed-bed approach, it is limited with respect to reliable quantification and/or enrichment of sample. A packed-bed approach is extremely difficult to apply in a manner that is both cost-effective and reliable. It cannot be effectively applied to a microscale process level.
Moreover, packed columns have extensive carry-over from sample to sample, are expensive to manufacture, and may be difficult to multiplex (extract multiple samples simultaneously). Proteins may be irreversibly adsorbed to the extraction phase or may be trapped by frits and other “dead zones” within the column making recovery of the proteins incomplete.
Other drawbacks include losses of materials due to unswept volumes leading to low recoveries and irreproducibility of results; dilution of materials due to large elution volumes applied in an attempt to minimize these selfsame unswept volumes; depending on implementation, requirements often to adhere to a flow “directionality” introducing limitations on full integration of sample processing; manufacturing difficulties and costs for micro- or nanoscale volume systems; and porosity of construction materials used in commercially available systems that cause severe loss of biomaterials.
Spin columns and pipette tip columns are disposable column technologies commonly used for processing samples. At present, most of these columns contain filters or frits. Conventional frits, porous discs used to contain the column beds, have significant dead volume. This leads to significant sample loss when very small sample volumes are separated.
One conventional method for making sample preparation devices involves first inserting a precut porous plug obtained from, for example, a fiberous glass or cellulose sheet, into the tip of a pipette. This is followed by the addition of loose particles and a second porous plug. The plugs serve to retain the particles in place in the pipette tip. However, the plugs also entrap excess liquid thereby creating dead space or volume (i.e., space not occupied by media or polymer that can lead to poor sample recovery, contamination such as by sample carry-over, etc.).
Current available methods are not well suited for the separation and recovery of very small volumes in the low microliter range.
Also, since the volume of the filter is often as large as the volume of the micro volume sample itself, the extraction or separation process or chromatography process is adversely affected due to the large volume of filter material through which the sample must pass.
In addition, the adsorption of biomolecules can be a problem. Since the concentration of biomolecules in micro volume samples is so small, the adsorption of biomolecules on the filter can result in significant loss of the total sample mass. The filter material may also absorb proteins or biomolecules from the sample, resulting in lower than desirable sample recovery. Also, the filter material may behave differently in different elution media, subsequently interfering with both the quality of the separation process and the volume of the sample retained.
Collecting samples in the 1 to 20 μL range is a critical need. At such low volumes, efficient sample handling is crucial to avoid loss. Conventional methods and devices for sample preparation are not practical for handling the “microseparation” of such small sample volumes.
Ultrafiltration can only effectively concentrate and desalt, and thus the application of adsorption technology at this scale could offer an entirely new approach to micro-mass sample preparation.
However, these procedures cannot be used with extremely small liquid delivery devices such as conventional pipette tips, as there is no practical way to load either the plug or the particles to obtain a micro-adsorptive device that contains 20 milligrams or less of adsorbent, the amount suitable for use with the aforementioned extremely small sample loads.